Alkane oxidation by modified hydroxylases

Abstract

This invention relates to modified hydroxylases. The invention further relates to cells expressing such modified hydroxylases and methods of producing hydroxylated alkanes by contacting a suitable substrate with such cells.

What is claimed is: 
     
       1. An isolated or recombinant polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 with up to 50 conservative amino acid substitutions excluding residues 47, 52, 74, 94, 142, 175, 184, 188, 205, 226, 236, 252, 255, 290, 353, 366 and 443 and comprising:
 (a) at position 82, a Z1 amino acid residue; 
 (b) at positions 78 and 328, a Z4 amino acid residue, and 
 wherein Z1 is an amino acid residue selected from the group consisting of glycine (G), asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), and cysteine (C); 
 wherein Z4 is an amino acid residue selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), and 
 wherein the polypeptide catalyzes the conversion of an alkane to an alcohol. 
 
     
     
       2. The polypeptide of  claim 1 , further comprising at position 464 of SEQ ID NO:1, an amino acid residue selected from the group consisting of tyrosine (Y), arginine (R), glycine (G), and threonine (T). 
     
     
       3. The polypeptide of  claim 2 , wherein at position 464 of SEQ ID NO:1 is a glycine (G). 
     
     
       4. The polypeptide of  claim 1 , wherein the up to 50 conservative amino acid substitutions comprises up to 25 amino acid substitutions. 
     
     
       5. The polypeptide of  claim 1 , wherein the up to 50 conservative amino acid substitutions comprises up to 10 amino acid substitutions. 
     
     
       6. The polypeptide of  claim 1 , wherein the up to 50 conservative amino acid substitutions comprises up to 5 amino acid substitutions. 
     
     
       7. The polypeptide of  claim 1 , wherein the alkane is selected from the group consisting of methane (CH 4 ), ethane (C 2 H 6 ), propane (C 3 H 8 ), butane (C 4 H 10 ), pentane (C 5 H 12 ), hexane (C 6 H 14 ), heptane (C 7 H 16 ), octane (C 8 H 18 ), nonane (C 9 H 20 ), decane (C 10 H 22 ), undecane (C 11 H 24 ), and dodecane (C 12 H 26 ). 
     
     
       8. The polypeptide of  claim 1 , wherein the alcohol is selected from the group consisting of methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, nonanol, decanol, undecanol, and dodecanol. 
     
     
       9. The polypeptide of  claim 1 , further comprising at position 710 of SEQ ID NO:1, a threonine (T). 
     
     
       10. The polypeptide of  claim 2 , further comprising at position 710 of SEQ ID NO:1, a threonine (T). 
     
     
       11. The polypeptide of  claim 10 , wherein the alkane is selected from the group consisting of methane (CH 4 ), ethane (C 2 H 6 ), propane (C 3 H 8 ), butane (C 4 H 10 ), pentane (C 5 H 12 ), hexane (C 6 H 14 ), heptane (C 7 H 16 ), octane (C 81 H 18 ), nonane (C 6 H 20 ), decane (C 10 H 22 ), undecane (C 11 H 24 ), and dodecane (C 12 H 26 ). 
     
     
       12. The polypeptide of  claim 10 , wherein the alcohol is selected from the group consisting of methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, nonanol, decanol, undecanol, and dodecanol.